Trimethoprim/sulfamethoxazole has historically been the treatment of choice for infection caused by Stenotrophomonas maltophilia. This study sought to define the pharmacodynamic indices and magnitude of exposure required for stasis and 1 log10 cfu reductions. Pharmacodynamic studies were conducted using the in vitro chemostat model over 24 h against three trimethoprim/sulfamethoxazole-susceptible S. maltophilia isolates with MICs from 0.25/4.75 to 2/38 mg/L. The primary endpoint was the change in cfu at 24 h relative to baseline. The log ratio of the area under the cfu curve (LR AUcfu) was a secondary endpoint. Trimethoprim and sulfamethoxazole exposures required for stasis and 1 log10 cfu/mL reduction were determined. Trimethoprim/sulfamethoxazole exposures achieved stasis and 1 log10 cfu/mL reductions in 9/16 (56%) and 2/16 (13%) of experiments. Both the fAUC/MIC and fCmax/MIC were identified as equivalent pharmacodynamic drivers, with stasis achieved at an fAUC/MIC of 67.4 and 30.0 for trimethoprim and sulfamethoxazole, respectively. Clinically meaningful exposures required to achieve 1 log10 cfu/mL reductions were not quantifiable. The LR AUcfu analysis supported the lack of overall bacterial burden reduction against S. maltophilia. In this in vitro chemostat model, trimethoprim/sulfamethoxazole monotherapy, even at higher doses, achieved limited activity against susceptible S. maltophilia.